Automated system for determining the molecular weight and/or concentration of macromolecules via sedimentation equilibrium

Abstract

An automated system for measuring the concentration gradient of centrifuged solute and directly calculating the molecular weight and/or sedimentation coefficient of an optically absorbing solute. Monochromatic light passes through a stationary slit and a previously centrifuged sample in a tube while the tube is moved vertically at a uniform rate by a platform which is driven by a motor and gearing, including a gear nut which drives a vertical screw attached to the platform. The transmitted light impinges on a photodetector which provides absorbance readings. This provides data of absorbance vs. time through the scanning cycle, which is processed digitally. A microcomputer is used to calculate the molecular weight and/or sedimentation coefficient from this data. The centrifuge tube is supported in a transparent cuvette containing a black plastic adaptor block which conformably receives the tube and supports it in upright position, allowing the light beam to pass through opposite vertical slot portions in the block. The cuvette rests on the platform, which is moved vertically relative to the slit defining the fixed beam path. A microswitch is operated at the upper limit of travel to stop the drive motor. The motor is external to the housing in which the optical components and the sample tube are located. The external motor is coupled by a flexible shaft to the drive gearing. Alternatively, a stepping motor may be employed instead of a synchronous motor to move the centrifuge tube in an incremental fashion rather than at uniform velocity.

What is claimed is: 
     
       1. A method of measuring the concentration gradient of solute in solution with a solvent in an optically transparent cylindrical centrifuge tube having a concentration gradient of solute along the cylindrical axis of said centrifuge tube, comprising (a) fitting said centrifuge tube, with said concentration gradient of solute therein, into a holder having a cylindrical bore, defined by a non-reflective bore surface, adapted for tightly fitting said centrifuge tube therein, an exterior shape having two parallel faces and a pair of vertical slits through said parallel faces of said exterior shape defining a transverse radiation passage including, when said centrifuge tube is fit into said cylindrical bore, said centrifuge tube;   (b) placing said holder, with said centrifuge tube therein, in an upright position into an optically transparent cuvette having flat parallel faces and an interior shape which is essentially the same as that of said exterior shape of said holder and being of an internal diameter slightly larger than that of said holder so as to securely position said holder in said cuvette interior;   (c) supporting the cuvette, with said holder and said centrifuge tube therein, in an upright position;   (d) passing a fixed beam of radiant energy transversely through the upright cuvette, with said holder and said centrifuge tube therein, along said transverse radiation passage;   (e) moving said cuvette, with said holder and said centrifuge tube therein, axially through a range of movement; and   (f) measuring the absorbancy of said fixed beam at a plurality of positions along said range of movement.   
     
     
       2. The method of claim 1, further comprising the step of filling space between said holder and said cuvette with a transparent liquid for reducing internal reflection and refraction of light passing through said cuvette and said transverse radiation passage, said transparent liquid contacting said centrifuge tube. 
     
     
       3. The method of claim 2, further comprising the steps of subjecting said centrifuge tube, when containing only said solvent therein, to said steps (a) through (f) and subtracting, for each of said plurality of positions, the absorbancy of said centrifuge tube with only said solvent therein from the corresponding absorbancy of said centrifuge tube with said solute concentration gradient therein, whereby an absorbancy valve corresponding only to said solute concentration gradient, unaffected by inhomogeneities in said centrifuge tube, is obtained. 
     
     
       4. The method of claim 2 wherein said centrifuge tube comprises quartz. 
     
     
       5. A method of determining the molecular weight of macro-molecular solute material comprising the steps of: (a) centrifuging said macro-molecular solute in a optically transparent cylindrical centrifuge tube substantially to a condition of sedimentation equilibrium;   (b) fitting said centrifuge tube, with said concentration of solute gradient therein, into a holder having a cylindrical bore defined by a non-reflective bore surface adapted for tightly fitting said centrifuge tube therein, an exterior shape having two flat parallel faces and a pair of vertical slits through said parallel faces of said exterior shape defining a transverse radiation passage including, when said centrifuge tube is fit into said cylindrical bore, said centrifuge tube;   (c) placing said holder with said centrifuge tube therein in an upright position into an optically transparent cuvette having flat parallel faces and an interior shape which is essentially the same as that of said exterior shape of said holder and being of an internal diameter slightly larger than that of said holder so as to securely position said holder in said cuvette;   (d) supporting the cuvette, with said holder and said centrifuge tube therein, in an upright position;   (e) passing a fixed beam of radiant energy transversely through the upright cuvette, with said holder and said centrifuge tube therein, along said transverse radiation passage;   (f) moving said cuvette with said holder and said centrifuge tube therein, axially through a range of movement; and moving said cuvette, with said holder and said centrifuge tube therein, axially through a range of movement, and;   (g) measuring the absorbancy of said fixed beam at a plurality of positions of the centrifuge tube along said range of movement, thereby enabling the determination of the gradient of solute concentration of the centrifuged solute material and the calculation of the molecular weight therefrom.   
     
     
       6. The method of claim 5, and wherein said fixed beam of radiant energy comprises monochromatic light. 
     
     
       7. The method of claim 5, and wherein the axial movement of the transparent container is at a uniform rate. 
     
     
       8. The method of claim 5, and halting the axial movement when the container reaches a predetermined limiting position relative to said beam. 
     
     
       9. The method of claim 5, wherein the container is supported in a vertical position and wherein said range of movement of the container is in a vertical direction. 
     
     
       10. The method of claim 5, and wherein said container is moved vertically through said plurality of positions in controlled increments. 
     
     
       11. The method of claim 5, and wherein said container is moved continuously through said range of movement. 
     
     
       12. The method of claim 5, and wherein the measured absorbance is recorded digitally at fixed time increments corresponding to said plurality of positions of the container. 
     
     
       13. Apparatus for measuring the concentration gradient of solute in a cylindrical, optically transparent centrifuge tube, formed by the prior application of a sedimenting force, comprising a holder having a cylindrical bore therein, said cylindrical bore being defined by a non-reflective bore surface and adapted for tightly fitting said cylindrical centrifuge tube therein, an exterior having flat parallel faces and a pair of vertical slits through said parallel faces of said exterior defining a transverse radiation passage, including the central axis of said cylindrical bore; an optically transparent cuvette having flat parallel faces and an interior shape which is essentially the same as that of the exterior shape of said holder and being of an internal diameter slightly larger than that of said holder; means to move said cuvette, with said holder having said centrifuge tube therein, axially through a predetermined range of movement;   a source of radiant energy to direct radiant energy from said source through said cuvette and along said transverse radiation passage; and   means to measure the absorbancy of said beam at a plurality of positions along said range of movement.   
     
     
       14. The apparatus of claim 13, and wherein said means to support said tube comprises transparent cuvette means shaped to conformably receive the tube and vertically movable platform means underlying said cuvette means. 
     
     
       15. The apparatus of claim 13, and wherein said means to support said tube comprises motor-driven vertically movable platform means underlying said cuvette means, and means to limit the vertical range of movement of said platform means. 
     
     
       16. The apparatus of claim 15, and wherein said platform means includes a vertical drive screw fixed thereto, driving gear means threadedly engaged with said drive screw, and an electric motor drivingly coupled to said driving gear means. 
     
     
       17. The apparatus of claim 13, and wherein said means to move the tube axially through said predetermined range of movement comprises platform means underlying said support means, vertical elevating means drivingly coupled to said platform means, an electric motor drivingly coupled to said elevating means, energizing circuit means connected to said motor, and limit switch means operable to deenergize said motor at a predetermined elevated position of said centrifuge tube. 
     
     
       18. The apparatus of claim 13, and wherein said source of radiant energy is monochromatic. 
     
     
       19. The apparatus of claim 13, and wherein said means to measure the absorbance comprises a photodetector aligned with said fixed path through the centrifuge tube, a microcomputer, and circuit means connecting the output of the photodetector to said microcomputer. 
     
     
       20. The apparatus of claim 13, and wherein said means to move said tube axially includes reversible, variable-speed motor means drivingly coupled to said means to support the tube in upright position. 
     
     
       21. The apparatus of claim 13, wherein said centrifuge tube is disposed within said cylindrical bore and said holder is disposed within said cuvette, and wherein space between said holder and said cuvette contains a transparent liquid for reducing internal reflection and refraction of light passing through said cuvette and said transverse radiation passage. 
     
     
       22. The apparatus of claim 21 wherein said exterior shape of the holder is rectangular. 
     
     
       23. The apparatus of claim 21, wherein said holder has vertically extending means for lifting said holder from inside said cuvette.